Pan-cancer investigation of psoriasis-related BUB1B gene: genetical alteration and oncogenic immunology

Unknown factors contribute to psoriasis' hyperproliferative, chronic, inflammatory, and arthritic features. Psoriasis patients have been linked to an increased risk of cancer, though the underlying genetics remain unknown. Since our prior research indicated that BUB1B contributes to the development of psoriasis, we designed and carried out this investigation using bioinformatics analysis. Using the TCGA database, we investigated the oncogenic function of BUB1B in 33 tumor types. To sum up, our work sheds light on BUB1B's function in pan-cancer from various perspectives, including its pertinent signaling pathways, mutation locations, and connection to immune cell infiltration. BUB1B was shown to have a non-negligible role in pan-cancer, which is connected to immunology, cancer stemness, and genetic alterations in a variety of cancer types. BUB1B is highly expressed in a variety of cancers and may serve as a prognostic marker. This study is anticipated to offer molecular details on the elevated cancer risk that psoriasis sufferers experience.

www.nature.com/scientificreports/ log2 function. We investigated the differences in BUB1B expression levels between both normal and cancer samples as well as paired tumors and adjacent normal tissues with R software (statistical packages car and stats) and tested the significance of differences with unpaired Wilcoxon crossover samples. The outcomes were displayed using ggplot2 R package 15 (https:// cran.r-proje ct. org/ web/ packa ges/ ggplo t2/ index. html).
Prognostic analysis. The TCGA prognostic profile was obtained from the UCSC database. All malignancies with fewer than ten samples were eliminated. The relationship between BUB1B expression and prognosis in multiple cancer species was investigated. The survivor R software package was used for proportional risk hypothesis testing (log-rank test) and fitted survival regression. The results of OS (overall survival), DSS (disease-specific survival), DFI (disease-free interval), and PFI (progression-free interval) analysis were presented as Kaplan-Meier curves and forest plots using the survminer and ggplot2 packages. The hazard ratios (HRs) and 95% confidence intervals were calculated using univariate survival analysis.
Genetic alteration analysis of BUB1B in pan-cancer. The cBioPortal database (http:// www. cbiop ortal. org/), which allows anybody to download, analyze, and view large-scale cancer genomic datasets for a range of malignancies, was used to uncover genetic modifications of BUB1B in pan-cancer. "TCGA Pan-Cancer Atlas Studies" was chosen as the cohort. Next, we typed "BUB1B" into the "Query" module. Alteration sites, types, and numbers for BUB1B could be found in the "cancer type summary" and "mutation" modules. Additionally, using the GDC database (https:// portal. gdc. cancer. gov/) and MuTect2 software 16 , we downloaded, processed, and integrated the Simple Nucleotide Variation dataset of all TCGA samples. The structural domain information of the protein was obtained from the R package maftools (version 2.2.10) 17 . The outcome was depicted using a lollipop plot. A chi-square test was also performed to examine the frequency of mutations in different cancer types in two groups that were separated by BUB1B expression level (high or low).
Correlation of BUB1B expression with immune cell infiltration and immune regulator genes. The infiltration score for each immune cell type was determined based on gene expression for each patient in each tumor using both the xCell 18 and Timer 19 methods of the R package IOBR 20 . The expression data for BUB1B and 60 marker genes from the inhibitory (24) and stimulatory (36) immune checkpoint pathways were also examined in each sample 21 . Spearman correlation between BUB1B and an inhibitory as well as a stimulatory immune checkpoint was determined (statistically significant was defined as p < 0.05). Recently, a method called ESTIMATE, which computes an immunological score based on certain gene expression patterns of immune cells, has been utilized to forecast the infiltration of non-tumor cells. In this study, an R package called ESTIMATE 22 (version 1.0.13) was taken to test stromal score, immune score as well as ESTIMATE score based on gene expression for every patient in each tumor type.
Correlation analysis between BUB1B expression and tumor stemness. We obtained tumor stemness scores including DNAs and RNAs for each tumor from prior studies 23 . After removing cancer species with fewer than 3 samples per tumor type, we ultimately got expression data for 37 cancer species. Then, we intersected BUB1B gene expression data with RNAss or DNAss to calculate the Spearman correlation coefficient. The results were visualized with a lollipop plot using the ggplot2 R package.
Gene set enrichment analysis. The top 100 BUB1B-correlated genes with the most comparable expression patterns to BUB1B were extracted from the TCGA datasets using the "Similar Gene Detection" module of GEPIA2 24 . Additionally, the STRING tool 25 (https:// string-db. org/) was taken to construct the BUB1B (Homo sapiens) organizational network with the basic parameters as followed: One of the active interaction sources is co-expression. The second active interaction source is evidence. There can be a maximum of 50 interactors. Medium confidence is the lowest required interaction score (0.40). GAGE 26 was used to analyze the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway data, and the ggplot2 R package was used to visualize the results. The KEGG database (http:// www. genome. jp/ kegg) provides annotation of the genes and pathways 27 . And p < 0.05 is the significance cutoff.

Results
Pan-cancer expression analysis of BUB1B. We found substantial upregulations of BUB1B in 33 tumors, as shown in www.nature.com/scientificreports/ Genetic alteration of BUB1B. The results showed that the alteration rate of BUB1B in pan-cancer was 2% of quired samples (Fig. 4A). Genetic alteration types and frequency of BUB1B showed differences in pan-cancer (Fig. 4B). The top 3 alteration frequencies were 7.02% in Uterine Carcinosarcoma, 6.24% in Uterine Corpus Endometrial Carcinoma, and 4.6% in Skin Cutaneous Melanoma, respectively. As shown in Fig. 4C, BUB1B was widely mutated across multiple cancer species, with the top 5 high-frequency mutations being Stomach and Esophageal carcinoma (STES), Uterine Corpus Endometrial Carcinoma (UCEC), Stomach adenocarcinoma (STAD), Esophageal carcinoma (ESCA), and Lung adenocarcinoma (LUAD) at 3.40%, 3.20%, 2.70%, 2.20%, and 1.70%. We used chi-square testing to assess differences in the frequency of gene mutations in the aforementioned 4 tumor types (UCEC, STAD, LUAD, ESCA), and the results were shown in Fig. 4D-G. TP53 and TTN were the top 2 mutated genes in STAD, LUAD, and ESCA. TTN mutation did not demonstrate a statistically significant difference between various expressed BUB1B groups in ESCA. TP53 mutations were more frequent in high-expressed BUB1B groups in STAD, LUAD, and ESCA. Additionally, the SCNA module of the TIMER was used to investigate the relationship between the SCNAs of BUB1B and the immune infiltration pattern. The results showed that SCNAs of BUB1B reduced immune cell enrichment in the aforementioned cancer types (Fig. 5), especially in STAD, indicating a clear link between BUB1B genetic alteration and immune cell infiltration enrichment.

Relationship of BUB1B expression and immunological environment in Pan-cancer.
Using the TIMER approach (Fig. 6A), we found that the expression of BUB1B was substantially associated with the quantity of invading immune cells: CD4+ T cells in 16 species, neutrophils in 19 species, B cells in 21 types, macrophages in 17 types, CD8+ T cells in 16 types, and DCs in 19 types. We discovered that the majority of the 38 immune cell subtypes are significantly linked with BUB1B expression in various tumor types using the deconvo xCell method. BUB1B expression was most strongly correlated with Th2 cells in various malignancies (Fig. 6B). Cancer patients' prognosis is affected by immunosurveillance, and tumors manipulate immunological checkpoints to elude immune responses. According to our results, the majority of immunomodulators found in LUSC, THYM, and NB were negatively associated with BUB1B expression. However, in GBMLGG, KIPAN, LIHC, and PAAD, the expression of BUB1B was positively linked with the majority of immune inhibitors, and immunostimulators (Fig. 6C).
Correlation analysis with the immune score using the ESTIMATE algorithm. We calculated immune infiltration scores for a total of 10,178 tumor samples from 44 tumor types, and Spearman's correlation coefficient was determined between the levels of immune infiltration and BUB1B expression using corr. test     www.nature.com/scientificreports/ leads to a bad prognosis. We ranked all of the engaged cancer kinds based on the absolute value of r in the three types of scores mentioned above, and the top five cancer types are presented in Fig. 7.
Correlation between BUB1B expression and tumor stemness. As cancer progresses, tumor cells may shed their differentiated phenotype and acquire progenitor and stem cell characteristics. Tumorigenic stemness can be assessed using RNA-derived mRNA expression (RNAss) and DNA-derived DNA methylation patterns (DNAss) 23 . The link between BUB1B expression and tumor stemness, as determined by RNAss and www.nature.com/scientificreports/ DNAss, was examined in the current study. The Spearman correlation for each tumor was evaluated, and BUB1B interacts with RNAss (Fig. 8A) and DNAss (Fig. 8B) to varied degrees. In particular, we discovered that 19 cancers (17 positives) had substantial correlations in DNAss, while 32 tumors (30 positives) had strong correlations in RNAss. Notably, BUB1B showed a positive association with DNA and RNA for several different forms of cancer types but showed a negative correlation with RNA for THYM. These contradictory findings imply that RNAss and DNAss may be able to distinguish between various malignant cell populations with varying characteristics or levels of stemness in various cancers.

BUB1B-related gene enrichment analysis.
To examine the functional mechanism of BUB1B in carcinogenesis, we gained the top 100 genes with similar expression patterns comparable to BUB1B from GEPIA2. The KEGG enrichment analysis revealed that the aforementioned 100 genes were strongly linked to the cell cycle or cellular senescence (Fig. 9A). The STRING tool was then used to gather 50 genes that co-expressed with BUB1B in order to validate the findings. The 50 genes were closely correlated, as seen in Fig. 9B, the genes were likewise involved in the cell cycle and cellular senescence (Fig. 9C). These findings lead us to hypothesize that BUB1B, through regulating the cell cycle and inducing cellular senescence, may contribute to tumorigenesis in malignancies.

Discussion
Several studies have been conducted on the high cancer risk in psoriasis patients, with the emphasis mostly on the effect of lifestyle (e.g., smoking and alcohol use) or treatment (e.g., PUVA or UVB irradiation and biologics) 2,28,29 . There are several factors that can contribute to cancer, but the most prevalent are exogenous factors and endogenous factors 30 . With the advent of genetic testing and the era of targeted therapy, molecular signatures have become increasingly important 31 . However, there is currently a scarcity of studies on the molecular basis. According to our prior research, BUB1B might be a gene that bridges the gap between psoriasis and cancer development 11 . Based on this, we sought to investigate the role of BUB1B in pan-cancer and provide potential molecular mechanisms for psoriasis and the concomitant high risk of cancer.  www.nature.com/scientificreports/ BUB1B is a critical component of the mitotic checkpoint, which is required for normal mitosis 32 . While BUB1B over-expression is reported in many different cancers, including breast cancer 33 , ECC 34 , PCa 35 , and so on, there has not been a comprehensive pan-cancer analysis of BUB1B' status. This study found that BUB1B was significantly upregulated in multiple tumors across both paired (18 tumor types) and unpaired (33 tumor types) sample analyses, which is consistent with the literature. On the basis of these results, we investigated the implications of differential BUB1B expression on prognosis, tumor immunity, and gene mutation in pan-cancer. Poor OS in 14 tumor types was revealed to be substantially linked with higher BUB1B expression using Cox and KM curve analysis (KIPAN, GBMLGG, LGG, KIRP, ACC, KICH, MESO, LIHC, KIRC, LUAD, PAAD, LAML, PCPG,  PRAD). Similar results were found in previous studies that high BUB1B expression was associated with poor prognosis in the three types mentioned above, suggesting that BUB1B is a viable prognostic factor for certain malignancies. A genetic alteration is an essential event in cancer development and progression. Moreover, the differences in the genetic molecular features of patients, such as DNA methylation, mutation, and copy number alterations (CNA), have been shown to be associated with clinical responses to anti-cancer drugs 36,37 . Given this, we investigated the genetic alteration of BUB1B in pan-cancer further. Stomach and Esophageal Carcinoma (STES), Uterine Corpus Endometrial Carcinoma (UCEC), Stomach Adenocarcinoma (STAD), Esophageal Carcinoma (ESCA), and Lung Adenocarcinoma (LUAD) had the highest mutation rates, with frequencies of 3.4%, 3.2%, 2.7%, 2.2%, and 1.7%, respectively. Somatic copy number alteration (SCNA) is an important form of somatic genetic alteration in cancer 38 . We then examined the association between the SCNAs of the BUB1B gene and immune infiltration in the aforementioned cancer types using the SCNA module of TIMER. The results showed that different SCNAs of BUB1B could decrease immune cell infiltration in aforementioned cancer types and have the greatest effect on STAD, suggesting that the genetic alteration of BUB1B is closely associated with immune cell infiltration.
However, the function of BUB1B and its impact on the tumor immune milieu have received little attention so far. Thus, we further investigated the relationship between BUB1B expression and tumor immune environment in pan-cancer. Using the TIMER method, it was revealed that BUB1B is significantly associated with B cell, CD8+ T cell, CD4+ T cell, macrophage, neutrophil, and DC infiltration levels in different cancers. Using the deconvo xCell approach, BUB1B expression was also found to be significantly associated with the majority of the 38 immune cell subtypes in diverse tumor types, especially Th2 cell (positive correlation), which demonstrate pro-cancer function via the secretion of interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) 39 . Traditionally, psoriasis has been thought to be a T helper type 1 (Th1)-dominated skin condition, but emerging research indicates that the Th1-Th2-Th17 balance is likely a crucial functional and genetic determinant of psoriasis [40][41][42] . The relationship between BUB1B and Th2 cells hasn't attracted attention yet, which may be a new research avenue. Utilizing the ESTIMATE algorithm, we discovered that high BUB1B expression lowered immune cell and stromal cell infiltration while increasing tumor cell quantity in 19 cancer types (GBM, UCEC, CESC, BRCA, ESCA, LUAD, STES, SARC, COAD, READ, STAD, HNSC, LUSC, TARGET-WT, SKCM, OV, BLCA, PCPG, ACC). The aforementioned impacts were most pronounced in STES, STAD, and LUSC. The present findings show a close relationship between BUB1B and the tumor immunological environment, implying that BUB1B could be a promising research and therapeutic target, however, further validation trials are required.
Growing evidence demonstrates that cancer stemness and immune evasion play a critical role in tumor development, progression, and metastasis 43 . Interestingly, a strong association between the expression of BUB1B and the majority of immunosuppressive as well as immunostimulatory molecules in various tumor types was also looked at in this study. Furthermore, in different cancer types, BUB1B demonstrated varying levels of connection with RNAss and DNAss (mainly positive association), implying that BUB1B may contribute to tumor progression by boosting cancer stemness. As a result, we hypothesize that increased BUB1B expression may promote cancer formation and potentially treatment resistance by boosting tumor stemness and assisting in immune surveillance evasion. To further uncover the possible signaling pathways involved in BUB1B in pan-cancer, we first obtained the top one hundred most similar gene sets to BUB1B using GEPIA and performed enrichment analysis on this gene set. The results showed that "cell cycle" was the most prominent, but "MicroRNAs in cancer", "Homologous recombination", "p53 signaling pathway", "FoxO signaling pathway" and "DNA replication" was also not negligible. This suggests BUB1B may contribute to cancer malignancy and progression through multiple mechanisms.
To summarize, our findings shed light on BUB1B's role in pan-cancer from a variety of angles, including pancancer expression, prognostic roles, genetic alterations, tumor stemness, and the possible correlation with tumor immune microenvironment, providing a theoretical foundation for the possibility of BUB1B as a pan-cancer marker. Yet, this study, which spanned various databases, had certain drawbacks. We performed a bioinformatic investigation of BUB1B, which made determining the value of clinical transformation challenging. Furthermore, because all of the databases' resources were tissue-derived, these findings cannot be confirmed with in vitro/in vivo models. Ultimately, further research into the mechanisms involved in carcinogenesis is needed.

Data availability
The datasets generated and analyzed during the current study are available in the UCSC database: UCSC Xena (xenabrowser.net). www.nature.com/scientificreports/